• The Korean Journal of Physiology and Pharmacology
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• 제20권2호
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• pp.169-175
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• 2016

Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) affect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a pro-inflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of inflammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL.

• 대한의생명과학회지
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• 제13권3호
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• pp.239-244
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• 2007

Hoxc8 is one of the homeotic developmental control genes regulating the expression of many downstream target genes, through which animal body pattern is established during embryonic development. In previous proteomics analysis, proliferating cell nuclear antigen (PCNA) which is also known as cyclin, has been implied to be regulated by Hoxc8 in F9 murine embryonic teratocarcinoma cell. When the 5' upstream region of PCNA was analyzed, it turned out to contain 20 Hox core binding sites (ATTA) in about 1.17 kbp (kilo base pairs) region ($-520{\sim}-1690$). In order to test whether this region is responsible for Hoxc8 regulation, the upstream 2.3 kbp fragment of PCNA was amplified through PCR and then cloned into the pGL3 basic vector containing a luciferase gene as a reporter. When the luciferase activity was measured in the presence of effector plasmid (pcDNA : c8) expressing murine Hoxc8, the PCNA promoter driven reporter activity was reduced. To confirm whether this reduction is due to the Hoxc8 protein, the siRNA against Hoxc8 (5'-GUA UCA GAC CUU GGA ACU A-3' and 5'-UAG UUC CAA GGU CUG AUA C-3') was prepared. Interestingly enough, siRNA treatment up regulated the luciferase activity which was down regulated by Hoxc8, indicating that Hoxc8 indeed regulates the expression of PCNA, in particular, down regulation in NIN3T3 cells. These results altogether indicate that Hoxc8 might orchestrate the pattern formation by regulating PCNA which is one of the important proteins involved in several processes such as DNA replication and methylation, chromatin remodeling, cell cycle regulation, differentiation, as well as programmed cell death.

• Parasites, Hosts and Diseases
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• 제51권1호
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• pp.61-68
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• 2013

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoebainduced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

• Animal cells and systems
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• 제15권2호
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• pp.139-146
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• 2011

Secretory leukocyte protease inhibitor (SLPI) plays an important role in promoting the invasion and metastasis of a range of cancer cells. However, there are no reports of the expression and function of SLPI in oral carcinoma cells. In this study, the oral carcinoma cell line KB was used to determine whether SLPI affects the proliferation, migration and invasion of oral carcinoma cells. RT-PCR and Western blotting revealed high levels of endogenous SLPI expression in KB cells as well as a strong increase in SLPI secretion after wounding compared to immortalized normal oral keratinocytes (INOK). The wound healing assay revealed more migration of KB cells than INOK cells, and the SLPI treatment increased the migration of KB cells. KB cell proliferation was increased significantly by the SLPI protein but decreased by SLPI-siRNA. SLPI strongly increased the migration and invasion of KB cells. On the other hand, SLPI-siRNA decreased the migration and invasion of KB cells. This suggests that SLPI plays an important role in the metastasis of oral carcinoma cells.

• BMB Reports
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• 제54권2호
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• pp.136-141
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• 2021

Thyroid eye disease (TED) is a complex autoimmune disease with a spectrum of signs. we previously reported that trisialoganglioside (GT)1b is significantly overexpressed in the orbital tissue of TED patients, and that exogenous GT1b strongly induced HA synthesis in orbital fibroblasts. However, the signaling pathway in GT1b-induced hyaluronic acid synthase (HAS) expression in orbital fibroblasts from TED patients have rarely been investigated. Here, we demonstrated that GT1b induced phosphorylation of Akt/mTOR in a dose-dependent manner in orbital fibroblasts from TED patients. Both co-treatment with a specific inhibitor for PI3K and siRNA knockdown of TLR2 attenuated GT1b-induced Akt phosphorylation. GT1b significantly induced HAS2 expression at both the transcriptional and translational level, which was suppressed by specific inhibitors of PI3K or Akt/mTOR, and by siRNA knockdown of TLR2. In conclusion, GT1b induced HAS2 in orbital fibroblasts from TED patients via activation of the PI3K-related signaling pathway, dependent on TLR2.

• 한국식품과학회지
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• 제32권6호
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• pp.1389-1395
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• 2000

자궁 경부암은 매년 약 50만명 정도씩 사망하는 여성의 치명적인 사망원인의 하나이다. 인두유종 바이러스(HPV) 16형 및 18형과 자궁 경부암과의 긴밀한 관련성은 잘 알려져 있다. 옻 추출물 Rhus가 HPV 16형의 E6, E7 발암 유전자를 억제하는지 여부를 측정하였다. 이 Rhus는 자궁 경부암 세포주(C-33A, SiHa, Caski)와 HaCaT keratinocytes의 분열은 농도 의존적으로 억제하였다. In vitro binding assay와 효소면역측정법에 의하면 Rhus가 암 억제인자인 p53과 결합하여 분해 시키는데 필수적인 E6와 E6AP와의 결합을 억제할 뿐더러 암 억제인자 Rb와 E7과의 결합을 억제하였다. RT-PCR에 의하면 Rhus에 의해 E6 mRNA의 level이 감소하였으나 E7 mRNA는 변하지 않았음을 보여주었다. 이들 결과에 의하면 Rhus가 HPV 16형의 E6와 E7의 발암성을 억제함을 보여 주므로 HPV에 의해 유도된 자궁 경부암의 치료에 유효할 것으로 사료되어 좀 더 자세한 in vitro실험 등이 요구된다.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1310-1317
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• 2018

The stems of Dendrobium moniliforme have been used in traditional herbal medicine for the treatment of fever and lack of body fluid in Korea. In this study, we investigated anti-inflammatory effects of the aqueous extract of D. moniliforme stems (DM) in response to lipoteichoic acid (LTA), a major constituent of the cell wall of Gram-positive bacteria. DM inhibited LTA-induced expression of a pro-inflammatory mediator inducible nitric oxide synthase (iNOS) in the murine macrophages. And DM induced expression of heme oxygenase-1 (HO-1) at the transcriptional level. Conversely, the knockdown of HO-1 expression by siRNA markedly reversed the inhibitory effects of DM on LTA-induced iNOS expression. We also demonstrated that nuclear translocation of Nrf2 was increased following treatment with DM. In addition, DM-mediated Nrf2 activation and HO-1 expression were suppressed by PI3K/Akt and p38 inhibitors; treatment with DM also resulted in phosphorylation of Akt and p38. These results suggest that DM inhibits the expression of iNOS in LTA-stimulated macrophages, and that these effects are mediated by the upregulation of HO-1 expression via PI3K/Akt/p38-Nrf2 signaling.

• Animal cells and systems
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• 제15권1호
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• pp.9-17
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• 2011

Colorectal cancer is the third leading cause of cancer-related death in Western countries. Chemotherapeutic agents with different mechanisms of action have shown an increase in cure rates. In the present study, we investigated the effect of a combination of low concentration of paclitaxel (taxol, 5 nM) and topoisomerase 1 inhibitor camptothecin (CPT) on HCT116 colon cancer cells. Although the viability of cells treated with taxol alone was similar to that of control cells, sequential treatment with taxol and CPT exhibited high cytotoxicity. However, the opposite sequence of treatment did not exert cytotoxic effects on HCT116 cells. This enhanced cytotoxicity of the sequential combination therapy was the result of mitotic arrest, which increased the level of $p21^{Cip1/WAF1}$ through the p38 mitogen-activated protein kinase (MAPK) pathway. Knockdown by $p21^{Cip1/WAF1}$ siRNA or treatment with a p38 inhibitor reduced the viability of cells sequentially exposed to taxol and CPT. Taken together, a low taxol concentration in combination with CPT induced mitotic arrest in HCT116 cells, leading to synergistic cell death through enhanced expression of $p21^{Cip1/WAF1}$ and p38 MAPK pathway. Therefore, taxol could playa role as a sensitizer of CPT in colon cancer cells.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.570-576
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• 2019

Particulate matter (PM), which refers to the mixture of particles present in the air, can have harmful effects. Damage to cells by PM, including disruption of organelles and proteins, can trigger autophagy, and the relationship between autophagy and PM has been well studied. However, the cellular regulators of PM-induced autophagy have not been well characterized, especially in keratinocytes. The Aryl Hydrocarbon Receptor (AhR) is expressed in the epidermis and is activated by PM. In this study, we investigated the role of the AhR in PM-induced autophagy in HaCaT cells. Our results showed that PM led to AhR activation in keratinocytes. Activation of the AhR-target gene CYP1A1 by PM was reduced by co-treatment with ${\alpha}$-naphthoflavone (${\alpha}-NF$), an AhR inhibitor. We also evaluated activation of the autophagy pathway in PM-treated keratinocytes. In HaCaT cells, treatment with PM treatment led to the induction of microtubules-associated proteins light chain 3 (LC3) and p62/SQSTM1, which are essential components of the autophagy pathway. To study the role of the AhR in mediating PM-induced autophagy, we treated cells with ${\alpha}-NF$ or used an siRNA against AhR. Expression of LC3-II induced by PM was decreased in a dose dependent manner by ${\alpha}-NF$. Furthermore, knockdown of AhR with siAhR diminished PM-induced expression of LC3-II and p62. Together, these results suggest that inhibition of the AhR decreases PM-induced autophagy. We confirmed these results using the autophagy-inhibitors BAF and 3-MA. Taken together, our results indicate that exposure to PM induces autophagy via the AhR in HaCaT keratinocytes.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.3781-3789
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• 2012

Background: Pancreatic cancer is one of the most aggressive tumors with a dismal prognosis. The membrane cytoskeletal crosslinker Ezrin participates in several functions including cell proliferation, adhesion, motility and survival. There is increasing evidence that Ezrin is overexpressed in vast majority of malignant tumors and regulates tumor progression. However, its roles in pancreatic cancer remain elusive. Methods: Three pairs of specific Ezrin siRNAs were designed and synthetized and screened to determine the most efficient one for construction of a hairpin RNA plasmid targeting Ezrin. After transfection into the Panc-1 pancreatic cancer cell line, real-time quantitative PCR and Western blotting were performed to examine the expression of mRNA and protein. The MTT method was applied to examine the proliferation and the drug sensibility to Gemcitabine. Flow cytometry was used to assess the cycle and apoptosis, while capacity for invasion was determined with transwell chambers. Furthermore, we detected phosphorylated-Erk1/2 protein and phosphorylated-Akt protein by Western blotting. Results: Real-time quantitative PCR and Western blotting revealed that Ezrin expression was notably down-regulated at both mRNA and protein levels by RNA interference (P< 0.01). Proliferation was inhibited and drug resistance to gemcitabine was improved (P< 0.05). Flow cytometry showed that the proportion of cells in the G1/G0 phase increased (P< 0.01), and in G2/M and S phases decreased (P< 0.05), with no apparent differences in apoptosis (P> 0.05). The capacity for invasion was markedly reduced (P< 0.01). In addition, down-regulating Ezrin expression had no effect on phosphorylated-Akt protein (P>0.05), but could decrease the level of phosphorylated-Erk1/2 protein (P< 0.05). Conclusions: RNA interference of Ezrin could inhibit its expression in the pancreatic cancer cells line Panc-1, leading to a potent suppression of malignant behavior in vitro. Assessment of potential as a target for pancreatic cancer treatment is clearly warranted.